Possible transsynaptic cholinergic neuromodulation by ATP released from ileal longitudinal muscles of guinea pigs

Life Sciences, Vol. 53, pp. 911-918 Printed in the USA

Pergamon Press

POSSIBLE TRANSSYNAPTICCHOLINERGIC NEUROMODULATIONBY ATP RELEASEDFROM ILEAL LONGITUDINALMUSCLESOF GUINEA PIGS

Takeshi Katsuragi 1, Katsuya Shirakabe, Osamu Soejima, Takeo Tokunaga, Katsuichi Matsuo, Chiemi Sato* & Tatsuo Furukawa Department of Pharmacology and *Research Laboratory of Biodynamics, School of Medicine, Fukuoka University, Fukuoka, 814-01, Japan (Received in final form June 28, 1993)

Summary

The effects of ~,B-methylene ATP (~,B-mATP) and B,y-methylene ATP (B,y-mATP) on endogenous acetylcholine (ACh) release evoked by electrical nerve stimulation were evaluated in guinea-pig ileal longitudinal muscles. Release of ACh was measured with an HPLCelectrochemical detector system and release of ATP by l u c i f e r i n luciferase assay. Electrically evoked endogenous ACh release was reduced by both ~,B-mATP and B,y-mATP at concentrations of 3 and 30 uM. The inhibitory effect of ~,B-mATP (30 uM) on ACh release was not detectable in the presence of theophylline (I00 ~M), a PIpurinoceptor antagonist, that i t s e l f enhanced ATP release. When exogenous ATP (O.l uM) was added to the bath in which the ileal segment was suspended, i t was rapidly metabolized, presumably by ecto-ATPase, and disappeared from the medium within 15 min. At 30 ~M, a,B-mATP induced ATP release in a suramin-sensitive but Ca2+and atropine-insensitive manner, suggesting P2-receptor-mediated release of ATP from the smooth muscle. We conclude from these findings that ~,B-mATP and, probably,also B,y-mATP, do not reduce ACh release by direct stimulation of presynaptic Pl-purinoceptors, and that endogenous ATP released postjunctionally by these ATP analogs is decomposed metabolically to adenosine in the synapse and this adenosine triggers Pl-purinoceptor sensitive neuromodulation of cholinergic transmission. Cholinergic neurotransmission is known to be modified by autoinhibition via activation of presynaptic muscarinic receptors ( I ) . In addition, there is evidence that purine compounds attenuate ACh release from cholinergic nerves through a presynaptic Pl-purinoceptor (2, 3, 4), which is much more responsive to adenosine than to ATP (5). However, i t has been reported that e l e c t r i c a l l y evoked ACh release is inhibited equally by ATP and adenosine and is antagonized by theophylline, a Pl-receptor antagonist (6, 7). Accordingly, i t is s t i l l controversial whether ATP per se or i t s breakdown product, adenosine, acts on presynaptic Pl-receptors for n~romodulation (8).

~,B-Methylene ATP (a,B-mATP) and B,y-methylene ATP (B,y-mATP), P2-receptor agonists, are ATP-analogs that are decomposed more slowly than ATP (9, I0). Moreover ~,B-mATP has been used as a desensitizing agent for the P2I. Author for correspondence. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.

912

Transsynaptic Neuromodulation of ACh Release

Vol. 53, No. 11, 1993

purinoceptor (II). There are r e p o r t s t h a t m,B-mATP causes t h e o p h y l l i n e s e n s i t i v e neuromodulation of c h o l i n e r g i c neurotransmission (9, 12), but the mechanism underlying the i n h i b i t o r y e f f e c t s of these d e g r a d a t i o n - r e s i s t a n t ATP analogs is s t i l l unknown. In the present work, we examined the nature of the presynaptic i n h i b i t i o n by ATP analogs of ACh release from the guinea-pig i l e a l l o n g i t u d i n a l muscle preparations, We also investigated the e f f e c t of the m,BmATP analog on p o s t j u n c t i o n a l release of ATP from these preparations.

Methods Male guinea-pigs (200 - 350 g) were stunned and bled and a d i s t a l portion of the ileum o f 3 cm l e n g t h was e x c i s e d . For p r e p a r a t i o n of l o n g i t u d i n a l muscle, the mucosal membranes or submucosal layers including c i r c u l a r muscles were c a r e f u l l y removed from the i l e a l muscles with f i n e tweezers and scissors under a microscope and the l o n g i t u d i n a l muscle s t r i p s (3 x 15 mm) were suspended in a bath of 3 ml of Krebs' solution (37°C) which was bubbled with 02 (95 %) plus CO2 (5 %). The composition of the Krebs' solution was as follows (in mM): NaCl, 122; KCl, 5.2; CaCI2, 2.4; MgSO4, 1.2; NaHCO3, 25.6; D-glucose, I I . 0 ; sodium-l-ascorbate, 0. I and Na2EDTA, 0.03. Measurement of endogenous ACh After loading with 0.5 g i n i t i a l tension, the preparation was allowed to equilibrate for 20 min. Physostigmine (3 ~M) was then added to the bath to protect released ACh from decay by acetylcholinesterase. After 30 min, 1 ml of the bathing solution was taken to measure ACh in the resting period (S-O). Then, I0 min l a t e r , e l e c t r i c a l stimulation (3 Hz, 0.3 msec, supramaximal voltage) with platinum electrodes was applied to the preparation repeatedly (55 sec-on and 5 sec-off) for 15 min. Samples of l ml of the bathing solution were taken 5 min (S-l), 10 min (S-2) and 15 min (S-3) after the start of electrical stimulation. Test drugs such as ~,B-mATP and B,y-mATP were added to the bathing medium from just after the f i r s t sampling (S-l) and were present during S-2 and S-3. Theophylline was introduced into the bath 20 min before starting electrical stimulation and was present until the end of the experiment. After every sampling, the same volume of bubbled Krebs' solution (37oc) containing the corresponding concentrations of physostigmine (3 ~M) and test drugs was added to the bath. Amounts of ACh in samples were collected at 5 min-intervals and were determined as reported previously (13). At the end of the experiment, the preparation was blotted with f i l t e r paper and weighed. The samples in tubes were frozen with 0. I ~M ethylhomocholine (a g i f t from Dr. Yamanishi, E i z a i , Tsukuba, Japan) as an i n t e r n a l s t a n d a r d , in a deep freezer. Then the ACh in the sample medium was concentrated by l y o p h i l i z i n g the s o l u t i o n in a f r e e z e - d r y e r w i t h a c o n c e n t r a t o r (Savant I n s t r u m e n t , H i c k s v i l l e , N.Y.) connected to a vacuum-pump. The residue was r e c o n s t i t u t e d in deionized water (I00 ~M), transferred to a microtube with a f i l t e r (pore size, 0.22 um; M i l l i p o r e Japan, Tokyo), and centrifuged. Amounts of endogenous ACh in aliquots (I0 ~I) were determined using an HPLC-electrochemical detector system (Yanagimoto Manufacturing Co., Kyoto, Japan)(13, 14). The detector p o t e n t i a l was set at +0.45V vs. an Ag/AgCl reference electrode. Analyses were carried out at a s e n s i t i v i t y ~ f 16 or 32 n A / f u l l scale. The mobile phase was prepared by a d d i n g 1 . 2 mM l - d e c a n e - s u l f o n a t e s o d i u m and 1 . 2 mM tetramethylammonium chloride as ion pair reagents (Nacalai, Kyoto) into I000 ml of 0. I M Na2HPO4.12H20 (pH 8.0) and degassed before use. The flow rate was 1.0 ml/min and the temperature was 32°C, throughout the measurement. Because 1 ml of ACh-free medium was added to the bath (3 ml) a f t e r c o l l e c t i n g samples at S-I and S-2, concentrations of ACh were d i l u t e d by 33.33 % and 66.67 %, r e s p e c t i v e l y . Thus, a correction was made for d i l u t i o n of ACh in the b a t h i n g medium a f t e r each sampling. Values f o r r e l e a s e d ACh were

Vol. 53, No. 11, 1993

Transsynaptic Neuromodulation of ACh Release

913

expressed as ~moles per g wet weight of tissue. Measurement of endogenous ATP L o n g i t u d i n a l muscle segments of ileum i s o l a t e d from g u i n e a - p i g s were prepared as described above and suspended in an organ bath (I ml) containing Krebs' s o l u t i o n (32°C) bubbled with 95 % 02 and 5 % CO2 throughout the experiment and were allowed to e q u i l i b r a t e for 30 min. Then, after washing with fresh solution, the segments were kept for 20 min in this solution. A c o n t r o l sample (50 ~ I ) was taken 5 min b e f o r e the end of the 50 m i n e q u i l i b r a t i o n period. A t e s t sample (50 ~ I ) was c o l l e c t e d 3 min a f t e r administration of a test drug, because the drug-evoked maximum contraction appeared w i t h i n 3 min. At the end of the experiment, the p r e p a r a t i o n was blotted with f i l t e r paper and weighed. Isometric tension of the preparation was monitored with a force transducer. For determination of endogenous ATP, sample media were treated with 250 ~I of ATP reagent s o l u t i o n (ATP bioluminescence HS, Boehringer Mannheim) containing l u c i f e r i n and luciferase in a glass microcell containing 200 ~I of deionized water. The l i g h t i n t e n s i t y produced a f t e r the reaction was measured by a luminometer (Lumicounter I000, Niti-on, Tokyo, Japan) equipped with a photomultiplier tube (15). Values of ATP release a f t e r exposure to test drugs are expressed as nmoles per g wet weight of tissue. ~,B-mATPper se at up to lO0 ~M did not react with luciferin-luciferase. S t a t i s t i c a l analysis Significances of differences between means and between multiple means were analyzed by Student's t - t e s t and by one way ANOVA followed by Dunnett's test, respectively. A P value of < 0.05 was regarded as s t a t i s t i c a l l y s i g n i f i c a n t . Drugs The drugs used were physostigmine hemisulfate, ~,B-methylene ATP (~,BmATP), B,y-methylene ATP (B,y-mATP), ~,B-methylene ADP (~,B-mADP), atropine sulfate (Sigma); ATP (Boehringer Mannheim); theophylline (Nacalai) and suramin (a g i f t from Bayer). All drugs were dissolved in de-ionized water before use. Results E f f e c t s o f ATP a n a l o g s on ACh r e l e a s e i n t h e p r e s e n c e and absence o f inhibitors ACh release from i l e a l l o n g i t u d i n a l muscles f o l l o w i n g e l e c t r i c a l stimulation (3Hz, 0.3 msec) (Table I) was t e t r o d o t o x i n - s e n s i t i v e (data not shown). The release was enhanced at S-I to S-3 in the presence of I00 RM theophylline, a Pl-purinoceptor antagonist. In c o n t r a s t , the evoked ACh release was decreased in the presence of ~,B-mATP or B,y-mATP in the S-2 and S-3 periods. Addition of m,B-mATP at 3 and 30 ~M reduced the release of ACh about 30 % and 36 % at S-2, and 33 % and 42 % at S-3. Similarly, B,y-mATP at 3 and 30 uM reduced the release about 14 % and 38 % at S-2, and 17 % and 40 % at S-3, respectively. Previous exposure to theophylline, however, almost completely prevented the r e d u c t i o n by a,B-mATP (30 ~M) of ACh release (TABLE I ). In the presence of 100 ~M suramin, a P2-purinoceptor antagonist (16, 17), the evoked-ACh release was decreased at S - I , but not at S-2 and S-3. The evoked-ACh release in the presence of suramin was no longer inhibited by ~,BmATP (30 ~M). Like suramin, a,B-mADP (30 ~M), an ecto 5 ' - n u c l e o t i d a s e i n h i b i t o r ( 1 8 ) , tended to decrease the ACh release at S-I without affecting contraction of the tissue. The i n h i b i t o r y e f f e c t of ~,B-mATP on the evoked ACh release was v i r t u a l l y abolished by these i n h i b i t o r s (TABLE I I ) .

914

Transsynaptic Neuromodulation of ACh Release

Vol. 53, No. 11, 1993

TABLE I E f f e c t s of ~,~-mATP and B,y-mATP on e l e c t r i c a l l y in the presence and absence of t h e o p h y l l i n e .

Treatment ( ~ M )

n

No treatment I0 ~,~-mATP ( 3) 5 ~,B-mATP (30) 6 B.y-mATP (3) 5 ~,~-mATP (30) 4 Theophylline(lO0) 5 Theophylline(lO0) 6 + ~,~-mATP ( 3 0 )

evoked-ACh release

ACh release (l~moles/g wet weight, mean ± S.E.) ..................................................... S-O S-I S-2 S-3 3.80 ± 0.50 6.77 ± 0.99 8.58 --6.00 . . . . 5.50 --7.32 . . . . . 5.28 5.78 ± 1.48 11.55 + 1.81" 14.08 . . . . . . 14.25

± I.I0 10.18 ± 1.38 ± 0.44* 6.77 ± 0.55* ± 0 . 2 2 * * 5.89 ± 0 . 5 5 * * ± 0.17 8.42 ± 0.66 ± 0 . 2 2 * * 6.05 ± 0 . 1 7 . * _+ 2.42* 17.60 ± 3 . 5 7 * * ± 0.93 a) 18.97 ± 0.55 a)

S-O, b a s a l r e l e a s e ; S - I , S-2 and S-3, e l e c t r i c a l l y - e v o k e d releases. Theophylline was a d m i n i s t e r e d I 0 min b e f o r e s t a r t i n g electrical s t i m u l a t i o n and was present u n t i l S-3. ~,~-mATP and B.y-mATP were added to the bath immediately a f t e r S-I sampling. * and * * ; s i g n i f i c a n t d i f f e r e n c e from t h e c o r r e s p o n d i n g c o n t r o l i(g?~ t r e a t m e n t ) a t P < 0.05 and P < 0.01 respectively, a) n o t s i g n i f ntly different from the value with t h e o p h y l l i n e alone.

TABLE I I E f f e c t s of ~,B-mATP on evoke-ACh release in the presence of ~.B-mADP or suramin. ACh release (umoles/g wet weight, mean ± S.E.) ..................................................... S-O S-I S-2 S-3

Treatment ( ~ M )

n

No treatment ~,B-mADP ( 3 0 ) ~.~-mADP + ~.B-mATP(30) Suramin (I00) Suramin + ~.B-mATP

6 5 5

2.70 ± 0.38 2.48 ± 0.50 ---

6.71 ± 0.33 5.06 ± 0.99

8.09 ± 0.61 8.97 ± 0.72 6.11 ± 1.49 7.04 ± I . I 0 5.50 ± 1.60 a) 6.44 ± 1.93 a)

4 4

2.26 ± 0.22

4.57 ± 0.82" ---

5.72 _+ 0.38 6.60 ± 0.38 4.90 ± 0.38 a) 5.68 _* 0.27 a)

~.~-mADP and suramin were administered in the same conditions as t h e o p h y l l i n e described in TABLE I. * ; s i g n i f i c a D t d i f f e r e n c e from corresponding control (no t r e a t m e n t ) at P < 0.05. a) not significantly different from corresponding c o n t r o l .

Vol. 53, No. 11, 1993

Transsynaptic Neuromodulation of ACh Release

915

Effect of ~,B-mATP on ATP release In longitudinal muscles of guinea-pig ileum, release of endogenous ATP accompanied with transient contraction was observed within 3 min after administration of m,B-mATP of concentrations of 10 and 30 ~M. The ATP release evoked by 30 uM ~,B-mATP was v i r t u a l l y unaffected by exposure to 0.3 ~M atropine, but was reduced in the presence of 300 ~M suramin, suggesting that the ATP released was myogenic in origin and the release was mediated by P2purinoceptors (TABLE I l l ) . TABLE I I I

Effects of ~,B-mATP on ATP release in the presence and absence of atropine or suramin. Treatment

(~M)

~,B-mATP (I0) ~,B-mATP (30) Ca2+-free + ~,B-mATP (30) Atropine (0.3) + ~,B-mATP (30) Suramin (300) + m,B-mATP (30)

Net ATP release above the basal level (nmoles/g wet weight, mean ± S.E.) (n)

P value

8.7 ± 0.6 27.4 ± 2.1 29.8 ± 4.5

(5) (7) (8)

n.s.

25.0 ± 2.4

(4)

n.s.

2.4 ± 0,2

(4)

< 0.05 a)

The basal ATP release was 15.0 ± 2.3 nmoles/g wet weight (n=6). ATP release was measured 3 min after addition of ~,B-mATP. Atropine and suramin were administered 15 min before adding ~,B-mATP. CaZ+-free s o l u t i o n w ~ prepared by adding 1 mM EGTA to Ca2+-free Krebs' solution, s i g n i f i c a n t l y d i f f e r e n t from value with ~,B-mATP (30 ~M) alone.

Ecto-ATPase a c t i v i t y in i l e a l segments Degradation of exogenously applied ATP in the bathing solution with and without i l e a l longitudinal muscle segment was measured over a 15 min period (FIG. I ) . When ATP (0.I ~M) was added to the bath without a longitudinal muscle segment, the amount of the n u c l e o t i d e in the medium remained v i r t u a l l y unchanged from the c o n t r o l (0 min) f o r 15 min. However, in medium with a suspended i l e a l segment, the amount of ATP in the bathing medium decreased quickly in a time-dependent manner to zero after 15 min, r e f l e c t i n g the fact that the i l e a l segment contains ecto-nucleotidase a c t i v i t y , probably ectoATPase.

Transsynaptic Neuromodulation of ACh Release

916

Vol. 53, No. 11, 1993

100-

m

o t"

° 50o

o~

O-

•

-1

I

0

!

I

I

I

5

I

!

I

Time FIG.

(mln)

I

I

10

i

I

!

I

|

15

1

Decay curves of ATP added e x o g e n o u s l y . Values are expressed as p e r c e n t a g e (mean ± S . E . ) of the c o n t r o l amount of ATP in medium sampled immediately a f t e r a d d i t i o n of the nucleotide (0. I ~M) to bathing medium with ( • , n=4) and without ( 0 , n=4) a suspended i l e a l segment. *; s i g n i f i c a n t d i f f e r e n c e from the c o r r e s p o n d i n g c o n t r o l (P < 0 . 0 1 ) . Values at - I min i n d i c a t e amounts of ATP in control medium.

Discussion

There have been a v a r i e t y of i n t e r p r e t a t i o n s of the underlying mechanisms of regulation of neurotransmissions by stable ATP analogs such as ~,B-mATP. The present work using an HPLC-electrochemical d e t e c t o r for determination of ACh revealed that e l e c t r i c a l l y evoked ACh release from guinea pig i l e a l longitudinal muscles was c l e a r l y reduced by ~,B-mATP or B,y-mATP. The r e d u c t i o n w i t h ~,~-mATP was a b o l i s h e d in the presence of t h e o p h y l l i n e . I n t e r e s t i n g l y , t h e o p h y l l i n e alone enhanced basal and evoked release of ACh from the segments by some as yet unknown mechanism. Furthermore, the i n h i b i t o r y e f f e c t of ~,~-mATP was not observed f o l l o w i n g exposure to 30 ~M ~,~-mADP and I00 ~M suramin, although ~,B-mADP and suramin per se tended to decrease the evoked ACh release by some unknown mechanism, According to von K~gelgen et al. (19), e l e c t r i c a l l y - e v o k e d overflow of [3H]NE from mouse vas d e f e r e n s is reduced by ATP and i t s analogs in the f o l l o w i n g o r d e r of potency; ATPyS > ATP = adenosine > U T P . However, the inhibiting effects o f ATP and ATPyS on 3 H - o v e r f l o w are n o t a f f e c t e d appreciably by 8 - ( p - s u l p h o p h e n y l ) t h e o p h y l l i n e (a Pl-antagonist), ~,B-ATP (a d e s e n s i t i z e r of P2x-receptors) and r e a c t i v e - b l u e 2 (a P v-antagonist, 20). Therefore, they concluded that the action of the purine 2-nr] c l e o t i d e s at the presynaptic s i t e is mediated via a type of P2-purinoceptor, presumably, a reactive blue 2 - r e s i s t a n t " P 2 y - l i k e " receptor. In t h e i r recent study, the

Vol. 53, No. 11, 1993

Transsynaptic Neuromodulation of ACh Release

917

evoked-release of [3H]NE from rabbit brain cortex slices was inhibited by adenosine and adenine nucleotides such as B,y-mATP, the order of potency being adenosine > ATP ATPyS B,y-imido ATP ADP > B,y-mATP. the i n h i b i t i o n s were markedly antagonized by 8-(p-sulphophenyl)theophylline, but not by suramin, implying that the nucleotide as well as adenosine acts on the Al-subtype of the Pl-purinoceptors (8), In the rat caudal artery or vas deferens, electrically-evoked NE release was decreased by purine compounds in the potency order of 2-chloroadenosine > B,y-mATP > ATP > adenosine. The i n h i b i t o r y effects were strongly antagonized by 8 - ( p - s u l p h o p h e n y l ) t h e o p h y l l i n e , but they were v i r t u a l l y unaffected by dipyridamole, an adenosine uptake i n h i b i t o r (21, 22). From these findings, i t has been postulated that a P3-purinoceptor may be present on the presynaptic sites. In contrast to adenosine and ATP, the potent P2x-receptor agonist ~,8mATP, enhanced [3H]ACh release from longitudinal muscle s t r i p s of guinea-pig ileum (23), i n d i c a t i n g t h a t the enhancement arose from s t i m u l a t i o n of presynaptic P2x-purinoceptors. In another experiment in which we measured ATP release from endotheliumf r e e l o n d i t u d i n a l muscles of the ileum d i r e c t l y , ~,B-mATP at 3 and 30 ~M produced suramin-sensitive, but Ca2+- or atropine-insensitive ATP release, thus, implying that the nucleotide is released from postjunctional sites of the tissue as reported previously (15, 24). Like ~,B-mATP, B,y-mATP has been shown to cause ATP release from i l e a l smooth muscles, although neither a,8-mADP nor adenosine affects the ATP release (24). On the basis of our r e s u l t s , we propose the f o l l o w i n g mechanisms to explain presynaptic neuromodulation by stable ATP analogs: Because they are chemically stable, ~,B-mATP and, probably also B,y-mATP are u n l i k e l y undergo enzymatic degradation to adenosine to cause neuromodulation of presynaptic PIpurinoceptors. T h u s ~,8-mATP, and probably also B,y-mATP, does not cause reduction of ACh release by direct stimulation of presynaptic Pl-purinoceptors. Endogenous ATP liberated p o s t j u n c t i o n a l l y by simulation of P2x-purinoceptors by these ATP analogs is decomposed metabolically to adenosine in the synapses and this adenosine triggers presynaptic Pl-purinoceptor-sensitive neuromodulation of cholinergic transmission (9), namely, trans-synaptic neuromodulation (25). This hypothesis gives some suggestion of the mechanisms of theophylline sensitive neuromodulations by P2-agonists, but further studies are required on this problem using more specific P2-antagonists or 5'-nucleotidase i n h i b i t o r s .

Acknowledgment This work was supported by a Grant-in-Aid for S c i e n t i f i c Research (No. 02670102) from the M i n i s t r y of Education, Science and Culture of Japan and a grant from the Suzuken Memorial Foundation (1990). We thank Mrs. Elizabeth A. Ichihara for careful reading of the manuscript and Bayer Japan for a generous g i f t of suramin.

References I. 2. 3. 4.

H. KILBINGER and A. STEIN, Br. J. Pharmacol. 94 1270-1274 (1988). K. TAKAGI and I. TAKAYANAGI, Jpn. J. Pharmacol. 22 33-36 (1972). L. GUSTAFSSON, P. HEDQVlST, B.B. FREDHOLM and G. LUNDGREN, Acta Physiol. Scand. 104 469-478 (1978). T° KATSURAGI, K. FURUTA, T. HARADA and T. FURUK.AWA, Gen. Pharmacol. 16 281-284 (1985a).

918

5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Transsynaptic Neuromodulation of ACh Release

Vol. 53, No. 11, 1993

G. BURNSTOCK, Cell membrane r e c e p t o r s f o r d f u ~ s and hormones: A multidisciplinary approac h. R.W. Staub and L. Bolis (eds), 107-118, Raven Press, New York (1978). E.S. VIZI and J. KNOLL. Neuroscience 1 391-398 (1976). T. KATSURAGI, K. FURUTA, T. HARADAand T. FURUKAWA, Clin. Exp. Pharmacol. Physiol. 12 73-78 (1985b). I. VON K-~-GELGEN, L. SPXTH and K. STARKE, Naunyn-Schmiedeb. Arch. Pharmacol. 346 187-196 (1992). C.J. MOODYand G. BURNSTOCK, Eur. J. Pharmacol. 77 I-9 (1982). N.J. CUSACK, J.D. PEARSONand J.L. GORDON, Biochem. J. 214 975-981 (1983). G. BURNSTOCKand C. KENNEDY, Gen. Pharmacol. 16 433-440 (1985). N.P. WIKLUND, L.Eo GUSTAFSSONand J. LUNDIN, Acta Physiol. Scand. 125 681691 (1985). T. KATSURAGI. K. SHIRAKABE, S. OGAWA, O. SOEJIMA, and T. FURUKAWA, J. Neurochem. 55 363-369 (1990a). P.E. POTTER, J.L. MEEKand N.H. NEFF, J. Neurochem. 41 188-194 (1983). T. KATSURAGI, T. TOKUNAGA, S. USUNE and T. FURUKAWA, Life Sci. 46 13011307 (1990b). P.M. DUNN and A.G.H. BLAKELEY, Br. J. Pharmacol. 93 243-245 (1988). C.H.V. HOYLE, G.E. KNIGHT and G. BURNSTOCK, Br. J. Pharmacol. 99 617-621 (1990). , W. SCHUTZ, J. SCHRADER and E. GERLACH, Am. J. Physiol. 240 H963-H970 (1981). I. VON K~GELGEN, E. SCH~FFEL and K. STARKE, Naunyn-Schmiedeb. Arch. Pharmacol. 340 522-532 (1989). G. BURNSTOCKand J . J . l . WARLAND, Br. J. Pharmacol. 90 383-391 (1987). K. SHINOZUKA, R.A. BJUR and D.P. WESTFALL, Naunyn-Schmiedeb. Arch. Pharmacol. 338 221-227 (1988). K.M. FORSYTH, R.A. BJUR and D.P. WESTFALL, J. Pharmacol. Exp. Ther. 256 821-826 (1991). B. SPERLAGHand E.S. VIZI, J. Neurochem. 56 1466-1470 (1991). T. KATSURAGI, T. TOKUNAGA, S. OGAWA, O. SOEJIMA, C. SATO and T. FURUKAWA. J. Pharmacol. Exp. Ther. 259 513-518 (1991). K. SHINOZUKA, K.O. SEDAA, R.A. BJUR and D.P. WESTFALL, Eur. J. Pharmacol. 192 431-434 (1991).